II. Overview of Laboratory Methods in Molecular Systematics

Review: structure and replication of DNA:

DNA is a polymer composed of four nucleotides.

Each nucleotide includes a pentose (five-carbon) sugar called deoxyribose (ribose in RNA), a nitrogenous base (adenine, cytosine, guanine, thymine), and a phosphate group, which is attached to the 5' carbon.

Polynucleotides include nucleotides linked by covalent bonds between the phosphate group and 3' carbon of adjacent nucleotides--single-stranded DNA molecules have polarity, with a 5’ end and a 3’ end.

DNA occurs in a double-stranded form called a duplex or double helix.

Double-stranded DNA molecules are held together by hydrogen bonds between nitrogenous bases of opposing nucleotides; A-T (with 2 H-bonds), C-G (3 H-bonds). These are relatively weak bonds—therefore DNA can be denatured with heat.

Opposing strands of DNA are complementary and have opposite polarity (they are antiparallel).

Single-stranded DNA molecules can anneal even if they do not have perfect complementarity—the greater the sequence similarity, the more stable the duplex (i.e., the more heat required to denature).

DNA is replicated by DNA poymerase, which synthesizes a complementary strand of DNA from a single-stranded template, following Watson-Crick base pairing rules.

DNA polymerase requires that the template be primed, with a short stretch of double-stranded DNA or DNA-RNA with an exposed 3' end.

DNA polymerase adds deoxyribonucleoside triphosphates (dNTP) to the 3’ carbon of the primer sequence, releasing pyrophosphate in the process—the 3’ carbon must have a hydroxyl group present for DNA polymerase to be able to add a new nucleotide.

The Polymerase Chain Reaction (PCR):

uses DNA polymerase to "amplify" genes

heat-stable Taq DNA polymerases (from thermophilic prokaryote, Thermus aquaticus) and automated thermal cycling machines make it possible to perform PCR quickly in a single tube.

A PCR reaction contains:

template DNA
primer 1
primer 2
dNTPs (dATP, dCTP, dGTP, dTTP)
buffer
TaqDNA polymerase

A typical PCR reaction cycles through a temperature regime, such as:

1. 2 min 95C (initial denaturation)
2. 1 min 94C
3. 30 sec 50C (annealing primers to template--annealing temp. is critical)
4. 1 min 72C (extension at optimal temperature for Taq polymerase)
5. go to 2 for 30 cycles
6. 5 min 72C (final extension)
7. 4C indefinite (stops reaction, hold until ready to be removed from thermal cycler)

PCRrequires knowledge of sequences flanking region of interest to design primers, which are single-stranded synthetic DNA molecules, typically 15-25 bp long.

rDNA has lots of known primer sites in conserved regions.

PCR is very sensitive to contamination from non-target DNAs, especially other PCR products.

DNA sequencing--traditional Sanger method (1977):

also uses DNA polymerases to generate copies of DNA from a primed template

takes advantage of fact that DNA polymerases can add dideoxynucleotides to growing chain, but cannot add nucleotides to a ddNTP that has been incorporated--hence, this is called the "chain termination" method.

fragments terminated with ddATP will end at sites corresponding to "T" in template; fragments terminated with ddCTP end at sites corresponding to "G" in template, and so on...

traditional method uses DNA polymerase derived from E. coli, and radioactively labeled nucleotides to detect fragments.

A traditional Sanger sequencing uses four reaction mixes, each containing:

template DNA
primer 1 with radioactive label
dNTPs (dATP, dCTP, dGTP, dTTP)
one of four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP)
DNA polymerase
buffer

sequencing reaction is incubated at 37C (optimal temperature for E. coli DNA polymerase) then quick frozen to stop reaction

each of the four reactions is loaded onto a single lane of an acrylamide gel (i.e., four lanes per template), and electrophoresed

in electrophoresis, DNA fragments, or other macromolecules, are separated by size as in a gel matrix under electric current.

acrylamide gel separates fragments that differ by a single base pair

after gel has run for an appropriate length of time, it is removed, dried, and exposed to x-ray film, which is developed to reveal positions of fragments

sequence of bases can be read as a "ladder" of bands in 4 lanes

Advances in DNA sequencing I: cycle sequencing:

E. coli DNA polymerase was replaced by Taq polymerase

allowed multiple cycles of annealing/synthesis/denaturation--thus increasing the quantity of sequencing products

reactions performed at higher temperatures allowed resolution of artifacts caused by secondary structure (folding and base-pairing) in templates

Advances in DNA sequencing II: fluorescent automated sequencing (1986):

radioactive label replaced by fluorescent label, bonded to ddNTPs--i.e., labeling step and chain termination step are combined

each unique ddNTP has a unique fluorophore (fluorescent dye), which emits a characteristic wavelength of light when illuminated by a UV laser.

fluorescent automated sequencing uses one reaction mix containing:

template DNA
primer 1
dNTPs (dATP, dCTP, dGTP, dTTP)
fluorescently labeled ddNTPs (ddATP*, ddCTP*, ddGTP*, ddTTP*)
Taq DNA polymerase
buffer

sequencing reaction is loaded on a single lane of an acrylamide gel

laser and photodetector scans across one line in the gel, automatically recording wavelengths of light emitted by DNA fragments as they migrate past

computer automatically compiles composite image from individual scan lines

sequence of nucleotides in template is estimated from the sequence of colors in each lane

Other kinds of molecular characters

restriction fragment length polymorphism (RFLP), restriction mapping

Chromosomal rearrangements/fusions

Duplication of genes/genomes

Microsatellites

etc.

References:

Campbell, N. A., and J. B. Reece. 2002. Biology, Sixth Edition. Benjamin Cummings. See Chapter 25, Phylogeny and Systematics.

Freeman, S., and J. C. Heron. 2000. Evolutionary Analysis. Prentice Hall. Excellent introductory text for general evolutionary biology. Includes good coverage of molecular phylogenetic methods.

Hillis, D. M., C. Moritz, and B. K. Mable (eds.). Molecular Systematics, Second Edition. 1996. Sinauer Associates. Multiauthored text treats laboratory methods as well as analytical considerations. Chapter 11 by David Swofford (author of the PAUP* program) is a thorough review of phylogenetic methods. Now somewhat dated, but still an essential reference.

Hall, B. G. 2001. Phylogenetic trees made easy: A how-to manual for molecular biologists. Sinauer Associates. As the name implies, this is a cook-book aimed at non-specialists. Provides excellent step-by-step instructions on how to perform alignment and phylogenetic analysis, with adequate justification of the methods. However, there is minimal theoretical background. Very accessible. Includes lists of download sites for freeware.

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